This invention relates to a method of screening for compounds that modulate gene expression, particularly those which lower gene expression, and compositions useful in such methods.
Methods are known which lower or abolish gene expression. For example, gene knockouts may be performed to abolish gene expression. Conditional lethal mutants may also be created to abolish gene expression and identify essential genes (see, for example, de Lorenzo, V. et al., Gene 123:17-24 (1993); Neuwald, A. F. et al., Gene 125: 69-73(1993); and Takiff, H. E. et al., J. Bacteriol. 174:1544-1553(1992). Chemical mutagenesis is yet another way to make such mutants Beckwith, J., Methods in Enzymology 204: 3-18(1991). Ribozymes provide another way to lower gene expression levels by damaging the gene or transcript. It has also been reported that the hammerhead RZ with abasic residues can be activated by addition of the base in trans. A drawback to these methods of preparing gene knockouts or lowering gene expression is that they are often time consuming and difficult to reproduce.
A SELEX generated RNA aptamer that binds malachite green has been reported. This complex, when illuminated at 630 nm, results in cleavage of the RNA by free radical generation as shown by abolishing marker gene activity.
The present invention provides improved methods for modulating, lowering and/or abolishing gene expression in a sequence-directed manner.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
The invention provides a method for modulating gene expression comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting an organism with the binding site and binding agent; and detecting modulation of gene expression.
The invention also provides a preferred method, wherein after the contacting step the binding site and binding agent enter the cell and contact an RNA target or DNA target.
A further preferred method comprises the step of having the binding site base pair with an RNA target molecule.
The invention also provides a method wherein the modulation of gene expression is lowering or inhibiting gene expression.
Moreover, the invention provides a method wherein the binding site is selected from the group consisting of a site that binds: malachite green, a photoreactive dye, a free radical generating compound, an iron salt, an iron ion, a radioactive iron radionuclide, isofuran blue, an isosulfan blue derivative, reactive sulfonyl chloride of isosulfan blue, hypocrellins and hypericin, tetrabromorhodamine, Rose Bengal photooxidizing dye, a FotoFenton reagent, 2-mercaptopyridine N-oxide, phenanthrolines, a spontaneous nitric oxide donors, DEANO, spermine NONOate, S-nitrosoglutathione, SNAP, SIN-1, M-7904, 2-hydroxyacetophenone oxime and Fenton""s reagent.
Further, the invention provides a method wherein the binding site is selected from the group consisting of a site that: binds a metal ion, creates a coordination complex with a metal ion or other atom or molecule, and is an iron response element.
Also provided is a method wherein the polynucleotide binding agent is selected from the group consisting of: malachite green, a photoreactive dye, a free radical generating compound, an iron salt, an iron ion, a radioactive iron radionuclide, isofuran blue, an isosulfan blue derivative, reactive sulfonyl chloride of isosulfan blue, hypocrellins and hypericin, tetrabromorhodamine, Rose Bengal photooxidizing dye, a FotoFenton reagent, 2-mercaptopyridine N-oxide, phenanthrolines, a spontaneous nitric oxide donors, DEANO, spermine NONOate, S-nitrosoglutathione, SNAP, SIN-1, M-7904, 2-hydroxyacetophenone oxime and Fenton""s reagent.
A method is also provided for altering the structure of a polynucleotide comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting a polynucleotide with the binding site and binding agent; and detecting an alteration in the structure of the polynucleotide.
A method is provided wherein the alteration in the structure is a cleavage of the polynucleotide phosphate backbone.
Another method is provided for modulating the function of a polynucleotide comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting a polynucleotide with the binding site and binding agent; and detecting modulation of the function.
The invention provides a method for modulating gene expression comprising the steps of: selecting a polynucleotide binding agent binding site and polynucleotide binding agent; contacting an organism with the binding site and binding agent; irradiating the binding agent with electromagnetic radiation; and detecting modulation of gene expression.
A preferred method is also provided wherein the contacting step also comprises contacting the binding site agent with a metal ion, metal salt or metal oxide.
Another preferred method is provided wherein the polynucleotide binding agent is malachite green and the electromagnetic radiation has a wavelength of about 630 nanometers (herein xe2x80x9cnmxe2x80x9d).
A kit is provide by the invention comprising at least on compartment containing an isolated polynucleotide binding agent binding site and a polynucleotide binding agent.
A preferred embodiment of the invention is a kit wherein the polynucleotide binding agent is selected from the group consisting of: malachite green, a photoreactive dye, a free radical generating compound, an iron salt, an iron ion, a radioactive iron radionuclide, isofuran blue, an isosulfan blue derivative, reactive sulfonyl chloride of isosulfan blue, hypocrellins and hypericin, tetrabromorhodamine, Rose Bengal photooxidizing dye, a FotoFenton reagent, 2-mercaptopyridine N-oxide, phenanthrolines, a spontaneous nitric oxide donors, DEANO, spermine NONOate, S-nitrosoglutathione, SNAP, SIN-1, M-7904, 2-hydroxyacetophenone oxime and Fenton""s reagent.
Another preferred embodiment of the invention is a kit wherein the polynucleotide binding agent is malachite green.
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
xe2x80x9cHost cellxe2x80x9d is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence and includes, but is not limited to organisms as defined elsewhere herein.
xe2x80x9cFlanking sequencexe2x80x9d is a polynucleotide sequence on either side (5xe2x80x2 or 3xe2x80x2) of and fused to the PBA binding site that is complementary, in whole or part to a polynucleotide sequence in the target polynucleotide.
xe2x80x9cIsolatedxe2x80x9d means altered xe2x80x9cby the hand of manxe2x80x9d from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not xe2x80x9cisolated,xe2x80x9d but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is xe2x80x9cisolatedxe2x80x9d, as the term is employed herein.
xe2x80x9cOrganism(s)xe2x80x9d means a (i) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chiamydia, Borrelia and Mycoplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigellaflexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium pegrfingens, Clostridium tetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon, including but not limited to Achaebacter, and (iii) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus Saccharomyces, Kiuveromyces, or Candida, Coccidiodes, Histoplasma, Cryptococcus and Paracoccidioides.
xe2x80x9cPolynucleotide(s)xe2x80x9d generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. xe2x80x9cPolynucleotide(s)xe2x80x9d include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, xe2x80x9cpolynucleotidexe2x80x9d as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term xe2x80x9cpolynucleotide(s)xe2x80x9d also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are xe2x80x9cpolynucleotide(s)xe2x80x9d as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term xe2x80x9cpolynucleotide(s)xe2x80x9d as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. xe2x80x9cPolynucleotide(s)xe2x80x9d also embraces short polynucleotides often referred to as oligonucleotide(s).
xe2x80x9cPBA binding sitexe2x80x9d herein means a polynucleotide that binds a PBA and/or a target polynucleotide.
xe2x80x9cPolynucleotide Binding Agentxe2x80x9d and xe2x80x9cPBAxe2x80x9d herein means an element or compound that binds to a polynucleotide.
xe2x80x9cTarget polynucleotidexe2x80x9d as the term is used herein, is a polynucleotide selected for structural or functional alteration.